Abstract. Members of the TGF-/~ superfamily appear to modulate mesenchymal differentiation, including the processes of cartilage and bone formation. Nothing is yet known about the function of the TGF-#-related factor vgr-1, also called bone morphogenetic protein-6 (BMP-6), and only limited studies have been conducted on the most closely related factors BMP-5, osteogenic protein-1 (OP-1) or BMP-7, and OP-2. Because vgr-1 mRNA has been localized in hypertrophic cartilage, this factor may play a vital role in endochondral bone formation. We developed antibodies to vgr-1, and documented that vgr-1 protein was expressed in hypertrophic cartilage of mice. To further characterize the role of this protein in bone differentiation, we generated CHO cells that overexpressed recombinant murine vgr-1 protein. Western blot analysis documented that recombinant vgr-1 protein was secreted into the media and was proteolytically processed to yield the mature vgr-1 molecule. To assess the biological activity of recombinant vgr-1 in vivo, we introduced the vgr-l-expressing CHO cells directly into the subcutaneous tissue of athymic nude mice. CHO-vgr-1 cells produced localized tumors, and the continuous secretion of vgr-1 resulted in tumors with a strikingly different gross and histological appearance as compared to the parental CHO cells. The tumors of control CHO cells were hemorrhagic, necrotic, and friable, whereas the CHO-vgr-1 tumors were dense, firm, and fibrotic. In contrast with control CHO tumors, the nests of CHO-vgr-1 tumor cells were surrounded by extensive connective tissue, which contained large regions of cartilage and bone. Further analysis indicated that secretion of vgr-1 from the transfected CHO tumor cells induced the surrounding host mesenchymal cells to develop along the endochondral bone pathway. These findings suggest that endogenous vgr-1 acts as an osteoinductive factor during endochondral bone formation. URtN6 development, a variety of growth and differentiation factors influence cell proliferation, differentiation, and migration. Several secreted peptide growth factors have been shown to mediate these processes, exerting their activities locally in an autocrine or paracrine fashion (reviewed in reference 20). The TGF-B superfamily represents one group of such factors that is particularly important in modulating mesenchymal differentiation (reviewed in references 10, 41). This family of factors influences pluripotent progenitor cells to differentiate into fibroblasts, adipocytes, myoblasts, chondrocytes, or osteoblasts. Correctly coordinated differentiation of mesenchymal cells

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Adeno-associated virus (AAV) has many properties of an ideal vector for delivery of therapeutic genes into the myocardium. Previous studies in a mouse model of myocardial infarction showed that AAV serotype 1 (AAV1) is superior to AAV serotypes 2-5 to transfer genes into the myocardium by direct injection. Since vectors may behave differently in humans and because the human and the pig hearts resemble each other closely, we tested whether AAV1 is also superior to AAV2 in transferring genes into the pig myocardium. We also compared gene transduction efficiency between AAV vectors and plasmid. We injected CMVLacZ and CMVVEGF (vectors with the cytomegalovirus (CMV) promoter driving LacZ and VEGF gene expression) unpackaged or packaged in AAV serotypes 1 or 2 capsids into pig myocardium. Hearts were collected 3, 14 and 28 days after the injection. Gene expression was analyzed by real-time reverse-transcription polymerase chain reaction (RT-PCR) and histological staining. Capillaries and smooth muscle alpha-actin (SMA)-positive vessels were quantified. Potential lymphocyte infiltration at the injection sites was analyzed by immunostaining using specific antibodies. As in the mouse, AAV1 mediated better gene transduction than AAV2. Plasmid mediated minimal gene expression only. More capillaries and SMA-positive vessels were detected at AAV1CMVVEGF- and AAV2CMVVEGF-injected than AAV1CMVLacZ-injected sites. We did not detect inflammatory cell infiltration at the injection sites. In conclusion, by direct injection, AAV1 is more efficient than AAV2, and plasmid is inefficient in mediating gene transfer into the pig myocardium. AAV-mediated VEGF gene transfer can also induce neovascular formation in the pig myocardium.